Project Abstract Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders resulting from mutations in mitochondrial or nuclear encoded genes that cause failures in mitochondrial energetic and metabolic function which, in the most severe cases, will lead to death. Incidence rates of 1:5000 have been reported placing mitochondrial disorders as one of the most commonly inherited human diseases. To date, there are no effective treatments, and as such, represents an urgent medical need to develop new technologies and platforms to uncover novel therapeutic opportunities. Identification of specific targets and drugs that increase mitochondrial bioenergetics can be of therapeutic value to treat mitochondrial diseases. To address this goal, we performed an unbiased high-throughput chemical and complementary genome-wide CRISPR editing screen in trans-mitochondrial hybrid (cybrids) cells harboring a mutation in a mitochondrial encoded complex I subunit. The IBET 525762A bromodomain and extraterminal domain (IBET) inhibitor emerged as the most potent compound to enhance the oxidative phosphorylation (OXPHOS) capacity in these cells. IBET 525762A functionally targets bromodomain-containing protein 4 (Brd4) and its inhibition enhances oxidative phosphorylation genes, protein, and activity. Furthermore, Brd4 inhibition promotes human cybrid cell survival under high energetic demands and protects against galactose-induced cell death (a standard clinical assay to identify mitochondrial defects). To explore the mechanism detailing how Brd4 inhibition controls mitochondrial bioenergetics, a series of comprehensive biochemical and metabolic analysis will be performed. Two specific aims will be assessed. In the first aim, we would like to explore on a molecular and functional level how Brd4 controls mitochondrial gene expression programs in cybrid cells. We will map the occupancy and interacting partners of Brd4 to gain insights into this mechanism. Aim 2 will assess the cellular, metabolic, and bioenergetic effects of Brd4 inhibition in other human cybrid and patient-derived fibroblasts cells with diverse mitochondrial disorders. We will determine if IBET 525762A-mediated increases in the OXPHOS program can persist to other cellular models of mitochondrial disease. The results from the proposed studies will improve our understanding of how Brd4 inhibition restores mitochondrial energetic function in mitochondrial disease cellular models. This will lead to the development of effective therapies for patients with mitochondrial disorders.